Influence of Calcium, Iron, and pH on Phosphate Availability for Microbial Mineralization of Organic Chemicals

A study was conducted to determine some of the factors affecting the P requirement for the biodegradation of p-nitrophenol, phenol, and glucose by Pseudomonas and Corynebacterium strains. Mineralization of glucose was rapid and the Pseudomonas sp. grew extensively in solutions with 5 and 10 mM phosphate, but the rate and extent of degradation were low and the bacterial population never became abundant in media with 0.2 mM phosphate. Similar results were obtained with the Corynebacterium sp. growing in media containing p-nitrophenol or phenol and in solutions with a purified phosphate salt. The extent of growth of the Corynebacterium sp. was reduced with 2 or 10 mM phosphate in media containing high Fe concentrations. Ca at 5 mM but not 0.5 mM inhibited p-nitrophenol mineralization by the Corynebacterium sp. with phosphate concentrations from 0.2 to 5.0 mM. Phenol mineralization by the Pseudomonas sp. in medium with 0.2 mM phosphate was rapid at pH 5.2, but the bacteria had little or no activity at pH 8.0. In contrast, the activity was greater at pH 8.0 than at pH 5.2 when the culture contained 10 mM phosphate. These effects of pH were similar in media with 5 mM Ca or no added Ca. We conclude that the effect of P on bacterial degradation can be influenced by the pH and the concentrations of Fe and Ca.     

Development of a substrate-coupled biocatalytic process driven by an NADPH-dependent sorbose reductase from Candida albicans for the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate

A substrate-coupled biocatalytic process was developed based on the reactions catalyzed by an NADPH-dependent sorbose reductase (SOU1) from Candida albicans in which ethyl 4-chloro-3-oxobutanoate (COBE) was reduced to (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE], while NADPH was regenerated by the same enzyme via oxidation of sugar alcohols. (S)-CHBE yields of 1,140, 1,150, and 780?mM were obtained from 1,220?mM COBE when sorbitol, mannitol, and xylitol were used as co-substrates, respectively. Optimization of COBE and sorbitol proportions resulted in a maximum yield of (S)-CHBE (2,340?mM) from 2,500?mM COBE, and the enantiomeric excess was 99.6?%. The substrate-coupled system driven by SOU1 maintained a stable pH and a robust intracellular NADPH circulation; thus, pH adjustment and addition of extra coenzymes were unnecessary.     

Purification and properties of aldose reductase form Pachysolen tannophilus

Aldose reductase (EC 1.1.1.21) from Pachysolen tannophilus IFO 1007 was purified 15 fold from the crude enzyme in a yield of 0.9% by pH 5 treatment, protamine sulfate precipitate, ammonium sulfate fractionation, and G-100 gel chromatography. The purified enzyme was entirely homogeneous on disc gel electrophoresis. The optimum pH and temperature were 5–6 and 50°C, and it was stable at pH 6–8 and up to 35°C. Its activity was enhanced slightly by Na₂SO₄, glycylglycine, glutathione, and cysteine, and inhibited remarkably by SH inhibitors such as AgNO₃, HgCl₂, lead acetate and iodo-acetate. Its K_m values were determined ad follows: 0.97 mM for d-glyceraldehyde, 1.7 mM for dl-glyceraldehyde, 3.5 mM for d-erythrose, 12 mM for d-xylose, 18mM for l-arabinose, 25 mM for galactose, 33 mM for valeraldehyde, 33 mM for 2-deoxy-d-glucose, 50 mM for propionaldehyde, 67 mM for d-ribose, 200 mM for d-mannose, and 280 mM for acetaldehyde. The enzyme also reduced glucose, l-sorbose, butylaldehyde, and benzaldehyde. Its molecular weight was estimated to be 40,650 by sedimentation equilibrium, 40,000 by SDS polyacrylamide gel electrophoresis and 43,000 by Sephadex G-200 column chromatography.     

High-Efficiency Conversion of Pyruvate to Acetoin by Lactobacillus plantarum during pH-Controlled and Fed-Batch Fermentations

The influence of pH on the type and concentration of metabolites produced from pyruvate by Lactobacillus plantarum ATCC 8014 was examined in pH-controlled fermentors at pH values of 4.5 to 6.5. Specific growth rates, cell dry weights, and diacetyl concentrations were highest at pH 5.5, with values of 0.78 h⁻¹, 190 mg/liter, and 1.2 mM, respectively. While the conversion efficiency (millimoles of acetoin formed per millimoles of pyruvate utilized) was highest (94.6%) at pH 4.5, acetoin levels were similar (20 mM) between pH 4.5 and 5.5. Feeding stationary-phase cells exogenous pyruvate increased acetoin levels to 78 mM.     

Production of Solvents by Clostridium acetobutylicum Cultures Maintained at Neutral pH

The formation of acetone and n-butanol by Clostridium acetobutylicum NCIB 8052 (ATCC 824) was monitored in batch culture at 35°C in a glucose (2% [wt/vol]) minimal medium maintained throughout at either pH 5.0 or 7.0. At pH 5, good solvent production was obtained in the unsupplemented medium, although addition of acetate plus butyrate (10 mM each) caused solvent production to be initiated at a lower biomass concentration. At pH 7, although a purely acidogenic fermentation was maintained in the unsupplemented medium, low concentrations of acetone and n-butanol were produced when the glucose content of the medium was increased (to 4% [wt/vol]). Substantial solvent concentrations were, however, obtained at pH 7 in the 2% glucose medium supplemented with high concentrations of acetate plus butyrate (100 mM each, supplied as their potassium salts). Thus, C. acetobutylicum NCIB 8052, like C. beijerinckii VPI 13436, is able to produce solvents at neutral pH, although good yields are obtained only when adequately high concentrations of acetate and butyrate are supplied. Supplementation of the glucose minimal medium with propionate (20 mM) at pH 5 led to the production of some n-propanol as well as acetone and n-butanol; the final culture medium was virtually acid free. At pH 7, supplementation with propionate (150 mM) again led to the formation of n-propanol but also provoked production of some acetone and n-butanol, although in considerably smaller amounts than were obtained when the same basal medium had been fortified with acetate and butyrate at pH 7.     

Acid carboxypeptidase from a wood-deteriorating basidiomycete, Pycnoporus Sanguineus

An acid carboxypeptidase (EC 3.4.16.1) has been isolated from the culture filtrate of a wood-degrading Basidiomycete, Pycnoporus sanguineus and the molecular and enzymatic properties of the enzyme were determined. The extracellular acid carboxypeptidase was homogeneous on polyacrylamide gel electrophoresis at pH 9.4 and SDS-disc gel electrophoresis. The MWs as determined by gel filtration and SDS-gel electrophoresis were 50 000 and 54 000, respectively. The isoelectric point was pH 4.78 using electrofocusing. The purified enzyme had a pH optimum of 3.4, a K_m of 0.74 mM and a k_cat of 16/sec with benzyloxycarbonyl-l-glutamyl-l-tyrosine. The K_m and k_cat values for bradykinin at pH 3.4 and 30° were 2.0 mM and 25/sec. Values for angiotensin at pH 3.4 and 30° were 0.76 mM and 2.4/sec, respectively.     

pH and Peptide Supply Can Radically Alter Bacterial Populations and Short-Chain Fatty Acid Ratios within Microbial Communities from the Human Colon

The effects of changes in the gut environment upon the human colonic microbiota are poorly understood. The response of human fecal microbial communities from two donors to alterations in pH (5.5 or 6.5) and peptides (0.6 or 0.1%) was studied here in anaerobic continuous cultures supplied with a mixed carbohydrate source. Final butyrate concentrations were markedly higher at pH 5.5 (0.6% peptide mean, 24.9 mM; 0.1% peptide mean, 13.8 mM) than at pH 6.5 (0.6% peptide mean, 5.3 mM; 0.1% peptide mean, 7.6 mM). At pH 5.5 and 0.6% peptide input, a high butyrate production coincided with decreasing acetate concentrations. The highest propionate concentrations (mean, 20.6 mM) occurred at pH 6.5 and 0.6% peptide input. In parallel, major bacterial groups were monitored by using fluorescence in situ hybridization with a panel of specific 16S rRNA probes. Bacteroides levels increased from ca. 20 to 75% of total eubacteria after a shift from pH 5.5 to 6.5, at 0.6% peptide, coinciding with high propionate formation. Conversely, populations of the butyrate-producing Roseburia group were highest (11 to 19%) at pH 5.5 but fell at pH 6.5, a finding that correlates with butyrate formation. When tested in batch culture, three Bacteroides species grew well at pH 6.7 but poorly at pH 5.5, which is consistent with the behavior observed for the mixed community. Two Roseburia isolates grew equally well at pH 6.7 and 5.5. These findings suggest that a lowering of pH resulting from substrate fermentation in the colon may boost butyrate production and populations of butyrate-producing bacteria, while at the same time curtailing the growth of Bacteroides spp.     

Characteristics of α-glucan phosphorylase from Chlorella vulgaris

α-Glucan phosphorylase from Chlorella vulgaris has been partially purified. In the direction of glucan phosphorolysis the apparent K_m for Pi was ca 2.4 mM at pH 7.1. In the direction of glucan synthesis the K_m for G1P was ca 0.12 mM at pH 6.2. The enzymic activity was inhibited by physiological concentrations of ADP, ATP, ADPG and UDPG. In the direction of starch degradation in the presence of 2.4 mM Pi the I_0.5 values for ADP and ATP were ca 1.6 and 2.9 mM, respectively, while in the direction of synthesis in the presence of 0.12 mM G1P the values were ca 0.23 and 1.4 mM, respectively. The Hill plots for starch degradation showed n values of 2.2 for ADP and 2.2 for ATP and values of 1.5 and 1.2, respectively, for starch synthesis. Both ADPG and UDPG were linear competitive inhibitors either with respect to Pi or with respect to GIP. The K_i values for ADPG and UDPG in the direction of phosphorolysis were shown to be ca 0.11 and 0.51 mM, respectively, and those in the direction of synthesis 0.033 and 0.15 mM, respectively.     

Growth conditions of clostridium perfringens type B for production of toxins used to obtain veterinary vaccines

The diseases caused for Clostridium perfringens are generically called enterotoxemias because toxins produced in the intestine may be absorbed into the general circulation. C. perfringens type B, grown in batch fermentation, produced toxins used to obtain veterinary vaccines. Glucose in concentrations of 1.4–111.1 mM was used to define the culture medium. The minimum concentration for a satisfactory production of vaccines against clostridial diseases was 55.6 mM. Best results were brought forth by meat and casein peptones, both in the concentration 5.0 g l^?1 in combination with glucose and a culture pH maintained at 6.5 throughout the fermentation process. The production of lactic, acetic and propionic organic acids was observed. Ethanol was the metabolite produced in the highest concentration when cultures maintained steady pH of 6.5 with exception of cultures with initial glucose concentration of 1.4 mM, where the highest production was of propionic acid. Maximal cell concentration and the highest toxin title concomitantly low yield coefficient to organic acids and ethanol were obtained using basal medium containing 111.1 mM glucose under a controlled pH culture (pH) 6.5 in batch fermentations of C. perfringens type B. These data contribute to improve process for industrial toxin production allowing better condition to produce a toxoid vaccine.     

Activation and inhibition of spinach ribulose 1,5-bisphosphate carboxylase by 2-phosphoglycolate

Ribulose 1,5-bisphosphate carboxylase when activated by preincubation with 1 mM bicarbonate and 10 mM magnesium chloride can be further activated ca 20–500% by incubating with 2.5 mM phosphoglycolate depending upon the pH of the preincubation medium. The activation effects were seen only under specific preincubation conditions. The activation by phosphoglycolate was a slow reaction requiring ca 15 min for maximal effect. Even though magnesium was essential for phosphoglycolate activation, concentrations higher than 15 mM progressively inhibited the activation of the enzyme by phosphoglycolate. When added directly to the reaction mixture, phosphoglycolate was a potent inhibitor of the carboxylase activity. Even under preincubating conditions, phosphoglycolate showed slight inhibitory effect at 0.1 mM and activation was observed at concentrations higher than 0.5 mM. The K_A value for phosphoglycolate was 2.8 mM.     

Intracellular Conditions Required for Initiation of Solvent Production by Clostridium acetobutylicum

We investigated the intracellular physiological conditions associated with the induction of butanol-producing enzymes in Clostridium acetobutylicum. During the acidogenic phase of growth, the internal pH decreased in parallel with the decrease in the external pH, but the internal pH did not go below 5.5 throughout batch growth. Butanol was found to dissipate the proton motive force of fermenting C. acetobutylicum cells by decreasing the transmembrane pH gradient, whereas the membrane potential was affected only slightly. In growing cells, the switch from acid to solvent production occurred when the internal undissociated butyric acid concentration reached 13 mM and the total intracellular undissociated acid concentration (acetic plus butyric acids) was at least 40 to 45 mM. Similar values were obtained when cultures were supplemented with 50 mM butyric acid initially or when a phosphate-buffered medium was used instead of an acetate-buffered medium. To measure the induction of the enzymes involved in solvent synthesis, we determined the rates of conversion of butyrate to butanol in growing cells. The rate of butanol formation reached a maximum in the mid-solvent phase, when the butanol concentration was 50 mM. Although more solvent accumulated later, de novo enzyme synthesis decreased and then ceased.     

Studies on the NADPH oxidation by subcellular particles from phagocytosing polymorphonuclear leucocytes. Evidence for the involvement of three mechanisms

1. The NADPH-oxidizing activity of a 100 000 × g particulate fraction of the postnuclear supernatant obtained from guinea-pig phagocytosing polymorphonuclear leucocytes has been assayed by simultaneous determination of oxygen consumption, NADPH oxidation and O^?₂ generation at pH 5.5 and 7.0 and with 0.15 mM and 1 mM NADPH.2. The measurements of oxygen consumption and NADPH oxidation gave comparable results. The stoichiometry between the oxygen consumed and the NADPH oxidized was 1 : 1.3. A markedly lower enzymatic activity was observed, under all the experimental conditions used, when the O^?₂ generation assay was employed as compared to the assays of oxygen uptake and NADPH oxidation.4. The explanation of this difference came from the analysis of the effect of superoxide dismutase and of cytochrome c which removes O^?₂ formed during the oxidation of NADPH.5. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reaction at pH 5.5. The inhibition was higher with 1 mM NADPH than with 0.15 mM NADPH.6. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reaction at pH 7.0 with 1 mM NADPH but less than at pH 5.5 with 1 mM NADPH.7. The effect of superoxide dismutase at pH 7.0 with 0.15 mM NADPH was negligible.8. In all instances the inhibitory effect of cytochrome c was greater than that of superoxide dismutase.9. It was concluded that the NADPH-oxidizing reaction studied here is made up of three components: an enzymatic univalent reduction of O₂; an enzymatic, apparently non-univalent, O₂ reduction and a non-enzymatic chain reaction.10. These three components are variably and independently affected by the experimental conditions used. For example, the chain reaction is freely operative at pH 5.5 with 1 mM NADPH but is almost absent at pH 7.0 with 0.15 mM NADPH, whereas the univalent reduction of O₂ is optimal at pH 7.0 with 1 mM NADPH.     

Purification and characterization of a new glucose dehydrogenase from vegetative cells of Bacillus megaterium

A new type of glucose dehydrogenase was purified from vegetative cells of Bacillus megaterium IAM1030. The characteristics of the vegetative-cell enzyme were investigated and compared with the enzyme from sporulating cells of B. megaterium IWG3. They are very similar in the following points: molecular size (M_r 120,000), subunit composition (homo tetramer), pH-activity profile with an optimum pH at around 8, pH-stability profile with a stable pH range of 6.0–7.5 (at 25°C, for 30 min), substrate specificity (specific for d-glucose and 2-deoxy-d-glucose), and the affinity for glucose (a K_m value of 11–12 mM at pH 8.0, 2.5 mM NAD). They are a little different in the following points: slower mobility for the vegetative-cell enzyme in polyacrylamide-gel electrophoresis at pH 8, immunological determinants (some of them are common), and higher heat resistance for the vegetative-cell enzyme at pH 6.5. They are quite different in their affinity for NAD and NADP. The K_m values for NAD are 0.1 mM for the vegetative-cell enzyme and 1.0 mM for the spore enzyme, while the values for NADP are 7.1 mM for the vegetative-cell enzyme and 0.09 mM for the spore enzyme, at pH 8.0, 0.1 M d-glucose. These results suggest that B. megaterium has at least two types of glucose dehydrogenase.     

Extraction and purification of a lectin from small black kidney bean (Phaseolus vulgaris) using a reversed micellar system

Lectin from crude extract of small black kidney bean (Phaseolus vulgaris) was successfully extracted using the reversed micellar extraction (RME). The effects of water content of organic phase (W_o), ionic strength, pH, Aerosol-OT (AOT) concentration and extraction time on the forward extraction and the pH and ionic strength in the backward extraction were studied to optimize the extraction efficiency and purification factor. Forward extraction of lectin was found to be maximum after 15 min of contact using 50 mM AOT in organic phase with W_o 27 and 10 mM citrate-phosphate buffer at pH 5.5 containing 100 mM NaCl in the aqueous phase. Lectin was backward extracted into a fresh aqueous phase using sodium-phosphate buffer (10 mM, pH 7.0) containing 500 mM KCl. The overall yield of the process was 53.28% for protein recovery and 8.2-fold for purification factor. The efficiency of the process was confirmed by gel electrophoresis analysis.     

Influence of Medium Buffering Capacity on Inhibition of Saccharomyces cerevisiae Growth by Acetic and Lactic Acids

Acetic acid (167 mM) and lactic acid (548 mM) completely inhibited growth of Saccharomyces cerevisiae both in minimal medium and in media which contained supplements, such as yeast extract, corn steep powder, or a mixture of amino acids. However, the yeast grew when the pH of the medium containing acetic acid or lactic acid was adjusted to 4.5, even though the medium still contained the undissociated form of either acid at a concentration of 102 mM. The results indicated that the buffer pair formed when the pH was adjusted to 4.5 stabilized the pH of the medium by sequestering protons and by lessening the negative impact of the pH drop on yeast growth, and it also decreased the difference between the extracellular and intracellular pH values (ΔpH), the driving force for the intracellular accumulation of acid. Increasing the undissociated acetic acid concentration at pH 4.5 to 163 mM by raising the concentration of the total acid to 267 mM did not increase inhibition. It is suggested that this may be the direct result of decreased acidification of the cytosol because of the intracellular buffering by the buffer pair formed from the acid already accumulated. At a concentration of 102 mM undissociated acetic acid, the yeast grew to higher cell density at pH 3.0 than at pH 4.5, suggesting that it is the total concentration of acetic acid (104 mM at pH 3.0 and 167 mM at pH 4.5) that determines the extent of growth inhibition, not the concentration of undissociated acid alone.     

Effects of pH and Sugar on Acetoin Production from Citrate by Leuconostoc lactis

The relationship between acetoin production and citrate utilization in Leuconostoc lactis NCW1 was studied. In a complex medium the organism utilized citrate at neutral pH (initial pH, 6.3) and at acid pH (initial pH, 4.5) but produced nine times more acetoin at the latter pH. In resting cells the utilization of citrate was optimum at pH 5.3. Production of acetoin as a function of citrate utilization increased as the pH decreased, and at pH 4.3 all of the citrate utilized was recovered as acetoin. Glucose (10 mM) and lactose (10 mM) markedly stimulated citrate utilization but totally inhibited acetoin production in glucose- and lactose-grown cells. Addition of glucose to cells actively metabolizing citrate caused an immediate increase in citrate uptake and a reduction in the level of acetoin. The apparent K_m values of lactic dehydrogenase for pyruvate were 1.05, 0.25, and 0.15 mM at pH 7.5, 6.5, and 5.0, respectively. Several heterofermentation intermediates inhibited α-acetolactate synthetase and decarboxylase activities. The implications of these results in regulating acetoin formatin are discussed.     

Oxidative and Reductive Routes of Glycerol and Glucose Fermentation by Escherichia coli Batch Cultures and Their Regulation by Oxidizing and Reducing Reagents at Different pHs

Glycerol and glucose fermentation redox routes by Escherichia coli and their regulation by oxidizing and reducing reagents were investigated at different pHs. Cell growth was followed by decrease of pH and redox potential (E _h ). During glycerol utilization at pH 7.5 ?pH, the difference between initial and end pH, was lower compared with glucose fermentation. After 8 h growth, during glycerol utilization E _h dropped down to negative values (?150 mV) but during glucose fermentation it was positive (+50 mV). In case of glycerol H₂ was evolved at the middle log phase while during glucose fermentation H₂ was produced during early log phase. Furthermore, upon glycerol utilization, oxidizer potassium ferricyanide (1 mM) inhibited both cell growth and H₂ formation. Reducing reagents dl-dithiothreitol (3 mM) and dithionite (1 mM) inhibited growth but stimulated H₂ production. The findings point out the importance of reductive conditions for glycerol fermentation and H₂ production by E. coli.     

Purification and properties of isocitrate lyase from Candida brassicae E-17

Isocitrate lyase (EC 4.1.3.1) was purified from acetate-grown cells of Candida brassicae E-17, by ammonium sulfate fractionation and DEAE-cellulose and Sephadex G-200 gel filtration column chromatographies. The purified enzyme was electrophoretically homogeneous. The molecular weight of this enzyme was 290,000 by gel filtration, and it was composed of four identical subunits whose molecular weights were 71,000 each. The pH and temperature optima were 6.8 and 37°C, respectively. The enzyme was stable from pH 6.0 to 7.0. The enzyme was activated by Mg²⁺ and the maximum activity was obtained with a concentration of 8 mM Mg²⁺. The enzyme was also activated by Mn²⁺ and Ba²⁺. The activity of this enzyme was stimulated by reducing agents. The K_m values for dl-isocitrate were 1.5 mM in sodium phosphate buffer and 0.62 mM in imidazole-HCl buffer.     

Invertases in young and mature leaves of Citrus sinensis

The sucrose catabolic enzymes acid invertase (EC 3.2.1.26) and alkaline invertase (EC 3.2.1.27) were studied in young and mature Citrus sinensis leaf tissue. In young, expanding leaves (60 % final length) soluble acid invertase activity predominated, while soluble alkaline invertase activity predominated in mature leaves. The acid and alkaline invertase activities were separated on Sephadex G-200. The acid invertase had an M_r of approximately 60 000, pH maximum of 4.5 and apparent K_m of 3.3 mM sucrose. The alkaline invertase had an M_r of approximately 200 000, pH maxima of 6.8 and an apparent K_m of 20 mM sucrose. Alkaline invertase was strongly inhibited by 10 mM Tris while acid invertase was not. Possible physiological roles for the two invertases are discussed.